CN102645508A - Detection method for six-component costustoot preparation - Google Patents

Abstract

The invention provides a detection method for a six-component costustoot preparation. The six-component costustoot preparation comprises six medicinal components, including costustoot, Veronica eriogyne H.Winkl, Phyllanthus emblica, cardamom, pomegranate seed and Fructus piperis longi. According to the method, costustoot, Veronica eriogyne H.Winkl, Phyllanthus emblica, cardamom, pomegranate seed and Fructus piperis longi in the six-component costustoot preparation are detected by using a thin layer chromatography method. The detection method has the advantages of good repeatability and stability, convenience in operation, high precision, high specificity, clear spot developing and high degree of separation. Through building of a reliable quality detection method with high specificity, the quality of the six-component costustoot preparation can be controlled effectively so that the quality of the six-component costustoot preparation become stable, safe and controllable.

Description

The detection method of Six-element banksia rose preparation

Technical field

The present invention relates to a kind of detection method of drug combination preparation, relate in particular to a kind of detection method of Six-element banksia rose preparation, belong to Chinese medicine detection technique field.

Background technology

Six-element banksia rose ball is a Tibetan medicine, records in " the Sanitation Ministry medicine standard " Tibetan medicine fascicle, and standard is numbered WS3-BC-0283-95.This standard record content mainly comprises:

Prescription: banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g.

Method for making: above Six-element, be ground into fine powder, sieve, mixing is used water pill, and drying promptly gets.

Proterties: these article are the sepia water-bindered pill; The special fragrance of the tool banksia rose, it is bitter to distinguish the flavor of.

Differentiate: get these article 2g, add ethyl acetate 20ml, refluxed 30 minutes in 60～80 ℃ of water-baths, filter, filtrating is concentrated into about 2ml, as need testing solution; Other gets the banksia rose, each 0.5g of seed of pomegranate control medicinal material, shines medicinal material solution in pairs with legal system; Get the pipering reference substance again, the accurate title, decide, and adds methyl alcohol and process the solution that every ml contains pipering 1mg, as reference substance solution.According to thin-layered chromatography (57 pages of appendix) test, draw each 10 μ l of above-mentioned four kinds of solution, put on same silica gel g thin-layer plate; With cyclohexane-ethyl acetate (6: 2) is developping agent, launches, and takes out; Dry; Put under the ultraviolet lamp (365nm) and inspect, in the test sample chromatogram, with reference substance chromatogram relevant position on show the fluorescence spot of same color.Spray is with 5% vanillic aldehyde concentrated sulfuric acid solution, in the test sample chromatogram, with control medicinal material chromatogram relevant position on show the spot of same color.

Inspection: should meet each item regulation relevant under the pill item.

Function with cure mainly: antiemetic relieves the pain.Be used for the pain that " Baconic's wood cloth " causes, belch, abdominal distension, vomiting etc.

Usage and consumption: one time 5～6 ball, 3 times on the one.

Specification: the heavy 6.5g of per 10 balls.

Storage: airtight, put shady and cool dry place.

The banksia rose: record in " one one of Chinese pharmacopoeia version in 2010, the 57th page, these article are the dry root of feverfew banksia rose Aucklandia lappa decene..Autumn, two seasons of winter excavate, and remove silt and fibrous root, and segment big is cutd open into lobe in again, hits tertia after the drying.

BAXIAGA: be the dry aerial parts of the little brightly yellowish violet Corydalis racemosa of bloodroot (Thunb.) Pers, the full-bloom stage aerial part of gathering dries or segment is dried subsequent use.

Emblic: record in " one one of Chinese pharmacopoeia version in 2010, the 167th page, this strain Tibetan conventional crude drugs is the dry mature fruit of euphorbia plant emblic Phyllanthus emblica L..Winter to time spring gathers during fruit maturation, removes impurity, drying.

Cardamom: record in " one one of Chinese pharmacopoeia version in 2010, the 156th page, these article are the dry mature fruit of zingiberaceous plant Amomum cardamomum Amomum kravanh Pierre ex Gagnep. or amomum compactum Soland ex Maton Amomumcompactun Soland ex Maton.Be divided into " former cardamom " and " Indonesia's cardamom " by place of production difference.

The Bi roots of grass: record in " one one of Chinese pharmacopoeia version in 2010, the 219th page, these article are dry near maturation or the mature fruit cluster of Piperaceae plant Bi roots of grass Piper longum L..Fruit ear is gathered during by green blackening, removes impurity, dries.

Seed of pomegranate: record in first the 26th page of Ministry of Health of the People's Republic of China's Tibetan medicine ministerial standard (standard numbering: WS3-BC-0026-95).These article are the dry seed of Punicaceae plant pomegranate Punicagranatum L..Remove pericarp behind the fruit maturation autumn, dries.

Visible by foregoing; Thin layer discriminating to the banksia rose, seed of pomegranate, the Bi roots of grass in the former ministerial standard all adopts unified method for distilling and developping agent condition to carry out; Find through thin layer discrimination test repeatedly in the practice; The banksia rose still has and negative disturbs, the Rf value of seed of pomegranate is higher, Bi roots of grass need testing solution does not have any spot, and the repeatability and the specificity of standard are relatively poor, can't realize the controllability to Six-element banksia rose ball quality standard detecting method.

Summary of the invention

Therefore; The detection method that the purpose of this invention is to provide a kind of Six-element banksia rose preparation; This detection method has been improved the method for distilling and the developping agent of the banksia rose in the former ministerial standard, seed of pomegranate, the Bi roots of grass; Detection method after the improvement is compared with primary standard, and repeatability and specificity are stronger, meets easy, principle fast; Set up the specificity thin layer discrimination method of emblic in the Six-element banksia rose preparation.

In the preferred embodiment of the present invention; Further set up the content assaying method of the contained costunolide of the banksia rose and dehydrogenation costunolide in the Six-element banksia rose preparation; So that provide a kind of favorable reproducibility, specificity strong; Meet accurate, easy, sensitive, principle fast, can effectively control the quality of product, make its steady quality, safety controlled.

To above-mentioned purpose, technical scheme of the present invention is following:

A kind of detection method of Six-element banksia rose preparation, said Six-element banksia rose preparation comprise the banksia rose, BAXIAGA, emblic, cardamom, seed of pomegranate and six kinds of medicinal ingredients of the Bi roots of grass; It is characterized in that this method is differentiated the banksia rose, seed of pomegranate, emblic and/or the Bi roots of grass in the said Six-element banksia rose preparation through thin-layered chromatography; Wherein:

(A) discriminating of the said banksia rose and seed of pomegranate may further comprise the steps:

(1) get said Six-element banksia rose preparation, add ethyl acetate, ultrasonic Extraction concentrates the back as the banksia rose, seed of pomegranate need testing solution with the gained extract;

(2) will go up the step gained banksia rose, seed of pomegranate need testing solution point sample in silica gel thin-layer plate, be that developping agent launches to differentiate with cyclohexane-methylene chloride-ethyl acetate; And/or

(B) discriminating of the said emblic and the Bi roots of grass may further comprise the steps:

(1 ') got said Six-element banksia rose preparation, adds ethanol, and ultrasonic Extraction concentrates the back as emblic, Bi roots of grass need testing solution with the gained extract;

(2 ') will be gone up step gained emblic, Bi roots of grass need testing solution point sample in silica gel thin-layer plate, be that developping agent launches to differentiate with petroleum ether-ethyl acetate-formic acid.

Preferably, also comprise in the said step (1) and get the banksia rose, seed of pomegranate control medicinal material, process the banksia rose, seed of pomegranate control medicinal material solution respectively according to the compound method of the said banksia rose, seed of pomegranate need testing solution; Also comprising in the step (2) the banksia rose, seed of pomegranate control medicinal material solution point sample in silica gel thin-layer plate, is that developping agent launches with cyclohexane-methylene chloride-ethyl acetate; And/or

Also comprise in the said step (1 ') and get the emblic control medicinal material, add ethanol, ultrasonic Extraction concentrates the back as emblic control medicinal material solution with the gained extract; Get the pipering reference substance, add the methanol solution that methyl alcohol is processed pipering, as the pipering reference substance solution; Also comprising in the step (2 ') emblic control medicinal material solution, pipering reference substance solution point sample in silica gel thin-layer plate, is that developping agent launches with petroleum ether-ethyl acetate-formic acid.

Preferably, comprise also in prescription ratio and preparation technology in the said step (1) that preparation does not contain the negative sample of the banksia rose, seed of pomegranate respectively, and processes the banksia rose, seed of pomegranate negative sample solution according to the compound method of the said banksia rose, seed of pomegranate need testing solution; Also comprising in the step (2) the banksia rose, seed of pomegranate negative sample solution point sample in silica gel thin-layer plate, is that developping agent launches with cyclohexane-methylene chloride-ethyl acetate; And/or

Comprise also in the said step (1 ') that in prescription ratio and preparation technology preparation does not contain the negative sample of emblic, the Bi roots of grass respectively, and processes emblic, Bi roots of grass negative sample solution according to the compound method of said emblic, Bi roots of grass need testing solution; Also comprising in the step (2 ') emblic, Bi roots of grass negative sample solution point sample in silica gel thin-layer plate, is that developping agent launches with petroleum ether-ethyl acetate-formic acid.

Preferably, said step (1) is: get Six-element banksia rose preparation 0.5～5 weight portion, behind the porphyrize, add ethyl acetate 5～40 parts by volume, sonicated 10～60 minutes filters, and filtrating is concentrated into 0.5～5 parts by volume, as the banksia rose, seed of pomegranate need testing solution; Other gets each 0.1～3 weight portion of the banksia rose, seed of pomegranate control medicinal material, processes the banksia rose, seed of pomegranate control medicinal material solution respectively according to the preparation method of the said banksia rose, seed of pomegranate need testing solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the said banksia rose, seed of pomegranate need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL.

Preferably, said step (1) is: get Six-element banksia rose preparation 2 weight portions, behind the porphyrize, add ethyl acetate 20 parts by volume, sonicated 30 minutes filters, and filtrating is concentrated into 2 parts by volume, as the banksia rose, seed of pomegranate need testing solution; Other gets each 0.5 weight portion of the banksia rose, seed of pomegranate control medicinal material, processes the banksia rose, seed of pomegranate control medicinal material solution respectively according to the preparation method of the said banksia rose, seed of pomegranate need testing solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the said banksia rose, seed of pomegranate need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL.

Preferably, said step (2) is: " sample solution that makes in the step (1) is drawn in an appendix VI of Chinese pharmacopoeia version in 2010 B test according to thin-layered chromatography; It is put respectively on same silica gel g thin-layer plate, is developping agent with cyclohexane-methylene chloride-ethyl acetate, and thin layer plate is put in the expansion cylinder saturated; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution, and the colour developing of wind to spot is clear.

Preferably; Need testing solution in the sample solution of said absorption and negative sample solution are 5～20 μ l, control medicinal material solution is 1～10 μ l, and need testing solution in the sample solution of preferred said absorption and negative sample solution are 10 μ l, control medicinal material solution is 5 μ l; The volume ratio of said developping agent cyclohexane-methylene chloride-ethyl acetate is 8～22: 3～8: 0.1～1, and preferred volume ratio is 15: 5: 0.5; Said thin layer plate was put in the expansion cylinder saturated 0～40 minute, preferred saturated 20 minutes; Said vanillic aldehyde sulfuric acid solution percent weight in volume is that 5% (take by weighing 5 weight portion vanillic aldehydes, add 100 parts by volume sulfuric acid, stirring promptly gets.Weight portion/parts by volume=g/ml), it is clear to blow to the spot colour developing with hot blast.

Preferably, said step (1 ') is: get Six-element banksia rose preparation 0.5～5 weight portion, behind the porphyrize, add ethanol 5～40 parts by volume, sonicated 10～60 minutes filters, and filtrating is concentrated into 0.5～5 parts by volume, as emblic, Bi roots of grass need testing solution; Other gets emblic control medicinal material 0.1～3 weight portion, processes emblic control medicinal material solution according to the preparation method of said emblic, Bi roots of grass need testing solution; It is an amount of to get the pipering reference substance again, adds methyl alcohol and processes the solution that every 1ml contains pipering 0.5～3mg, as the pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of said emblic, Bi roots of grass need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL.

Preferably, said step (1 ') is: get Six-element banksia rose preparation 2 weight portions, behind the porphyrize, add ethanol 20 parts by volume, sonicated 30 minutes filters, and filtrating is concentrated into 2 parts by volume, as emblic, Bi roots of grass need testing solution; Other gets emblic control medicinal material 0.5 weight portion, processes emblic control medicinal material solution according to the preparation method of said emblic, Bi roots of grass need testing solution; It is an amount of to get the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 1mg, as the pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of said emblic, Bi roots of grass need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL.

Preferably, said step (2 ') is: " sample solution that makes in the step (1 ') is drawn in an appendix VI of Chinese pharmacopoeia version in 2010 B test, and it is put in same silica G F respectively according to thin-layered chromatography ₂₅₄On the thin layer plate, be developping agent with petroleum ether-ethyl acetate-formic acid, thin layer plate is put in the expansion cylinder saturated, launches, and takes out, and dries, and under the 254nm ultraviolet lamp, inspects.

Preferably, the sample solution of said absorption is 5～20 μ l, preferably is 10 μ l; The volume ratio of said petroleum ether-ethyl acetate-formic acid is 1～10: 2～6: 0.05～0.5, and preferred volume ratio is 6: 4: 0.25, the boiling range of said sherwood oil is 60～90 ℃; Said thin layer plate was put in the expansion cylinder saturated 0～40 minute, preferred saturated 20 minutes.

Preferably, said detection method further comprises the content with contained costunolide of the banksia rose and/or dehydro-in the said Six-element banksia rose of the high effective liquid chromatography for measuring preparation, wherein:

The step of said high effective liquid chromatography for measuring comprises:

(1 ") preparation banksia rose need testing solution, and costunolide and/or dehydro-reference substance solution;

Step gained banksia rose need testing solution will be gone up in (2 "), and costunolide and/or dehydro-reference substance solution are injected liquid chromatograph and measured.

Preferably, also comprise preparation banksia rose negative sample solution in the said step (1 "); Comprise also in the said step (2 ") that banksia rose negative sample solution is injected liquid chromatograph to be measured.

Preferably, the chromatographic condition of said high effective liquid chromatography for measuring is: use octadecylsilane chemically bonded silica to be filling agent; With volume ratio is that 60～70: 30～40 acetonitrile-water is a moving phase; The detection wavelength is 225 ± 2nm; Column temperature: 20～40 ℃, number of theoretical plate calculates by the costunolide peak and is not less than 3000.

Preferably, the chromatographic condition of said high effective liquid chromatography for measuring is: use octadecylsilane chemically bonded silica to be filling agent; With volume ratio is that 65: 35 acetonitrile-water is a moving phase; The detection wavelength is 225nm; Column temperature: 25 ℃, number of theoretical plate calculates by the costunolide peak and is not less than 3000.

Preferably, said step (1 ") is: get Six-element banksia rose preparation 0.1～3 weight portion, behind the porphyrize, put in the tool plug conical flask; accurate methyl alcohol 10～50 parts by volume that add, and claims decide weight, and at power 250W, sonicated is 15～45 minutes under the frequency 40kHz; put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methyl alcohol, shakes up; filtration, gets subsequent filtrate, as banksia rose need testing solution; Precision takes by weighing the costunolide reference substance in addition and the dehydro-reference substance is an amount of; Add methyl alcohol and process the mixed solution that every 1ml contains costunolide 20～140 μ g and dehydro-40～200 μ g, mix reference substance solution as costunolide and dehydro-; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and processes banksia rose negative sample solution by the compound method of said banksia rose need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL.

Preferably, said step (1 ") is: get Six-element banksia rose preparation 1 weight portion, behind the porphyrize, put in the tool plug conical flask; accurate methyl alcohol 25 parts by volume that add, and claims decide weight, and at power 250W, sonicated is 30 minutes under the frequency 40kHz; put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methyl alcohol, shakes up; filtration, gets subsequent filtrate, as banksia rose need testing solution; Precision takes by weighing the costunolide reference substance with an amount of with the dehydro-reference substance in addition; The accurate title, decide; Add methyl alcohol and process the mixed solution that every 1ml contains costunolide 40 μ g and dehydro-60 μ g, mix reference substance solution as costunolide and dehydro-; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and processes banksia rose negative sample solution by the compound method of said banksia rose need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL.

Preferably, said step (2 ") be: accurate respectively absorption need testing solution, reference substance solution, each 5～20 μ l of negative sample solution, preferred each 10 μ l inject liquid chromatograph, and mensuration promptly gets.

Preferably, the bulk drug of said Six-element banksia rose preparation composition comprises: the banksia rose 200 weight portions, BAXIAGA 360 weight portions, emblic 500 weight portions, cardamom 80 weight portions, seed of pomegranate 400 weight portions and the Bi roots of grass 100 weight portions.

Preferably, the preparation method of said Six-element banksia rose preparation is: said bulk drug is ground into fine powder, sieves, mixing adds an amount of pill of water, promptly gets pill; Or said bulk drug is ground into fine powder, and sieving, mixing by the pharmacy conventional method, adds conventional auxiliary material, processes the oral formulations of accepting clinically: granule, pill, capsule, tablet or powder.

Preferably, said Six-element banksia rose preparation is granule, pill, capsule, tablet or powder.

This Tibetan medicine Six-element banksia rose preparation has and stomach pain relieving, antiemetic effect.Be used for the upper abdomen severe pain that treatment " Baconic's wood cloth " (being equivalent to gastritis, digestive tract ulcer) causes, n and V, belch etc.

Detection method of the present invention has been carried out the thin layer Study on Identification to the banksia rose, seed of pomegranate, emblic and the Bi roots of grass in the prescription.The present invention has further set up contained costunolide of the banksia rose and dehydro-high performance liquid chromatography content assaying method.Advantages such as detection method of the present invention has reappearance, good stability, method of operating is easy, precision is high, specificity is strong, the spot colour developing is clear, degree of separation is good; The quality determining method reliable through method for building up, that specificity is strong can effectively be controlled the quality of Six-element banksia rose preparation, makes the quality of Six-element banksia rose preparation reach stable, safety is controlled.

Description of drawings

Below, specify embodiment of the present invention in conjunction with accompanying drawing, wherein:

Fig. 1 differentiates the thin-layer chromatogram of the banksia rose, seed of pomegranate, the Bi roots of grass for the ministerial standard discrimination method; 1-3 is a test sample among the figure, and 4 is banksia rose control medicinal material, and 5 is banksia rose negative sample, 6 seed of pomegranate control medicinal materials, and 7 is the seed of pomegranate negative sample, and 8 is the pipering reference substance, and 9 is Bi roots of grass negative sample.

Fig. 2 is the thin-layer chromatogram of the banksia rose of the present invention, seed of pomegranate; 1-3 is a test sample among the figure, and 4-5 is a banksia rose control medicinal material, and 6 is banksia rose negative sample, and 7-9 is a test sample, and 10-11 is the seed of pomegranate control medicinal material, and 12 is the seed of pomegranate negative sample.

Fig. 3 is the thin-layer chromatogram of emblic of the present invention, the Bi roots of grass; 1-4 is a test sample among the figure, and 5 is the emblic control medicinal material, and 6 is the emblic negative sample, and 7-10 is a test sample, and 11 is the pipering reference substance, and 12 is Bi roots of grass negative sample.

Fig. 4 is the chromatogram of contained costunolide of the banksia rose of the present invention and dehydro-; Wherein chromatogram A is costunolide and dehydro-reference substance; Chromatogram B is a banksia rose test sample; Chromatogram C is a banksia rose negative sample.

Fig. 5 is the peak area of costunolide according to the invention and the linear relationship chart of reference substance concentration.

Fig. 6 is the peak area of costunolide according to the invention and the linear relationship chart of reference substance concentration.

Embodiment

Following experimental example and embodiment just are used to explain the present invention rather than restriction the present invention.

Unless otherwise indicated, the used developping agent and the ratio of moving phase are volume ratio in the embodiment of the invention.

Experimental example 1: the thin layer of the banksia rose, seed of pomegranate, the Bi roots of grass is differentiated

1.1 differentiate the banksia rose, seed of pomegranate and the Bi roots of grass according to the ministerial standard discrimination method

The preparation of need testing solution: get said Six-element banksia rose preparation 2g, add ethyl acetate 20ml, refluxed 30 minutes in 60～80 ℃ of water-baths, filter, filtrating is concentrated into about 2ml, as need testing solution;

The preparation of control medicinal material solution: get the banksia rose, each 0.5g of seed of pomegranate control medicinal material, shine medicinal material solution in pairs with legal system;

The preparation of reference substance solution: it is an amount of to get the pipering reference substance, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 1mg, as reference substance solution;

The preparation of banksia rose negative sample solution: in the prescription ratio, preparation does not contain the negative sample of the banksia rose, and processes banksia rose negative sample solution according to the preparation method of above-mentioned need testing solution;

The preparation of seed of pomegranate negative sample solution: in the prescription ratio, preparation does not contain the negative sample of seed of pomegranate, and processes seed of pomegranate negative sample solution according to the preparation method of above-mentioned need testing solution;

The preparation of Bi roots of grass negative sample solution: in the prescription ratio, preparation does not contain the negative sample of the Bi roots of grass, and processes Bi roots of grass negative sample solution according to the preparation method of above-mentioned need testing solution.

" each 10 μ l of above-mentioned six kinds of solution are drawn in an appendix VI of Chinese pharmacopoeia version in 2010 B test, put on same silica gel g thin-layer plate according to thin-layered chromatography; With cyclohexane-ethyl acetate (6: 2) is developping agent, launches, and takes out; Dry; Put under the ultraviolet lamp (365nm) and inspect, in the test sample chromatogram, with reference substance chromatogram relevant position on show the fluorescence spot of same color.Spray is that 5% (take by weighing 5 weight portion vanillic aldehydes, add 100 parts by volume sulfuric acid, stirring promptly gets with percent weight in volume.The vanillic aldehyde concentrated sulfuric acid solution of weight portion/parts by volume=g/ml), in the test sample chromatogram, with control medicinal material chromatogram relevant position on show the spot of same color.The result sees shown in the accompanying drawing 1: the feminine gender of the banksia rose has interference; The Rf value of seed of pomegranate is too big; In the Bi roots of grass need testing solution with the corresponding position of reference substance chromatogram on immaculate; Repeat that the result is still undesirable several times; So in this detection method, only the banksia rose and seed of pomegranate have been adopted unified approach; And method for distilling and developping agent carried out improving and perfect, the Bi roots of grass has carried out the thin layer discriminating in addition.

1.2 the present invention differentiates the thin-layer chromatography shaker test of the banksia rose, seed of pomegranate

(1) instrument

Mortar, graduated cylinder, round-bottomed flask, reflux, evaporating dish, sample applicator, chromatography cylinder, spray bottle, analytical balance (Mei Teletuo benefit, model: XS205), Extraction by Ultrasound device (Shanghai High Kudos Science Instrument Co., Ltd., model: SK8200HP), silica gel g thin-layer plate (Haiyang Chemical Plant, Qingdao, specification: 200 * 200mm).

(2) control medicinal material

Banksia rose control medicinal material (lot number: 120921-200506), the seed of pomegranate control medicinal material (lot number: 121431-200501), all available from Nat'l Pharmaceutical & Biological Products Control Institute.

(3) reagent

Ethyl acetate, methylene chloride, ethanol, methyl alcohol, the concentrated sulphuric acid, vanillic aldehyde, benzene, ethyl formate, formic acid, cyclohexane, distilled water.

(4) method of inspection:

Extract choice of Solvent: adopt ethanol, ethyl acetate, methyl alcohol respectively for extracting solvent;

The selection of method for distilling: adopt ultrasonic Extraction and refluxing extraction respectively;

The selection of developping agent: adopting cyclohexane-ethyl acetate (6: 2), benzene-ethyl acetate (19: 1), cyclohexane-ethyl formate-formic acid (15: 5: 1), cyclohexane-methylene chloride-ethyl acetate (15: 5: 0.5) respectively is developping agent.

Select above solvent, method for distilling and the developping agent of extracting to make an experiment respectively, the result is following:

Under above condition, through repetition test, confirmed that finally the specificity thin layer discrimination method of the banksia rose, seed of pomegranate is following:

Get said Six-element banksia rose preparation 2g, porphyrize adds ethyl acetate 20ml, and ultrasonic Extraction 30 minutes filters, and filtrating is concentrated into about 2ml, as the banksia rose, seed of pomegranate need testing solution; Other gets the banksia rose, each 0.5g of seed of pomegranate control medicinal material, processes the banksia rose, seed of pomegranate control medicinal material solution with method; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate respectively, and processes the banksia rose, seed of pomegranate negative sample solution according to the preparation method of above-mentioned need testing solution." each 10 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 5 μ l are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put respectively on same silica G plate according to thin-layered chromatography; With cyclohexane-methylene chloride-ethyl acetate (15: 5: 0.5) is developping agent, and thin layer plate was put in the expansion cylinder saturated 20 minutes, launches; Take out, dry, spray is 5% vanillic aldehyde sulfuric acid solution with percent weight in volume; It is clear that hot blast blows to the spot colour developing, and the result sees shown in the accompanying drawing 2.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, and negative noiseless.This method is through many people test of many times operation, favorable reproducibility, and specificity is strong, meets easy, sensitive, principle fast, can be used as the detection method of the banksia rose, seed of pomegranate in the Six-element banksia rose preparation.

1.3 the present invention differentiates the thin-layer chromatography shaker test of emblic, the Bi roots of grass

(1) instrument

Mortar, graduated cylinder, round-bottomed flask, reflux, evaporating dish, sample applicator, chromatography cylinder, spray bottle, analytical balance (Mei Teletuo benefit, model: XS205), Extraction by Ultrasound device (Shanghai High Kudos Science Instrument Co., Ltd., model: SK8200HP), silica gel g thin-layer plate (Haiyang Chemical Plant, Qingdao, specification: 200 * 200mm).

(2) control medicinal material and reference substance

The emblic control medicinal material (lot number: 121289-200301), the pipering reference substance (lot number: 110775-200203), all available from Nat'l Pharmaceutical & Biological Products Control Institute.

(3) reagent

Sherwood oil (boiling range 60-90 ℃), ethyl acetate, acetone, methyl alcohol, ethanol, toluene, formic acid, normal hexane, cyclohexane, glacial acetic acid, distilled water.

(4) method of inspection:

Extract choice of Solvent: adopting percent by volume respectively is that 70% ethanol (is promptly got 70ml ethanol; Add 30ml distilled water; Stir promptly and get), ethanol, percent by volume is that 70% methyl alcohol (promptly get 70ml methyl alcohol, add 30ml distilled water, stir promptly get), methyl alcohol are for extracting solvent;

The selection of method for distilling: adopt extraction, heating reflux method behind ultrasonic Extraction, the alcohol extract respectively;

The selection of developping agent: (4: 5: 0.4: 0.6), sherwood oil (boiling range 60-90 ℃)-ethyl acetate-formic acid (6: 4: 0.25) was developping agent to adopt toluene-ethyl acetate-acetone (7: 2: 1), cyclohexane-ethyl acetate-acetone (7: 2: 1), normal hexane-ethyl acetate-glacial acetic acid-methyl alcohol respectively.

Select above extraction solvent, method for distilling, developping agent to make an experiment respectively, the result is following:

Under above condition, confirmed finally that through repetition test the specificity thin layer discrimination method of emblic, the Bi roots of grass is following:

Get said Six-element banksia rose preparation 2g, porphyrize adds ethanol 20ml, and ultrasonic Extraction 30 minutes filters, and filtrating is concentrated into about 2ml, as emblic, Bi roots of grass need testing solution; Other gets emblic control medicinal material 0.5g, processes emblic control medicinal material solution with method; It is an amount of that essence is got the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that 1ml contains pipering 1mg, as the pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass respectively, and processes emblic, Bi roots of grass negative sample solution according to the preparation method of above-mentioned need testing solution." each 10 μ l of above-mentioned four kinds of solution are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put in same silica G F respectively according to thin-layered chromatography ₂₅₄On the plate, be developping agent with sherwood oil (60～90 ℃)-ethyl acetate-formic acid (6: 4: 0.25), thin layer plate was put in the expansion cylinder saturated 20 minutes, launched, and took out, and dried, and put under the ultraviolet lamp (254nm) and inspected, and the result sees shown in the accompanying drawing 3.In the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color, and negative noiseless.This method is through many people test of many times operation, favorable reproducibility, and specificity is strong, meets easy, sensitive, principle fast, can be used as the detection method of emblic, the Bi roots of grass in the Six-element banksia rose preparation.

Experimental example 2: the assay of contained costunolide of the banksia rose and dehydro-

(1) instrument

Conical flask, volumetric flask, transfer pipet, syringe, beaker, funnel, filter paper, filter, electronic balance (Mei Teletuo benefit, model: XS205), high performance liquid chromatograph (U.S. Agilent company, model: Aglient 1100).

(2) reference substance: costunolide (lot number: 111524-201006), dehydro-(lot number: 111525-200907), all identify institute available from Chinese pharmaceutical biological product.

(3) reagent: methyl alcohol, acetonitrile, ethanol, water.

(4) method of inspection:

Extract choice of Solvent: adopt methyl alcohol, ethanol respectively;

The selection of extraction time: adopt respectively to make an experiment in ultrasonic 20 minutes, 30 minutes, 45 minutes;

The selection of moving phase: adopt acetonitrile-water (65: 35), acetonitrile-water (60: 40), methanol-water (65: 35) respectively.

Select above solvent, extraction time and the moving phase extracted to make an experiment respectively, the result is as shown in the table:

Repetition test under above condition, confirmed that finally the method for the contained costunolide of the banksia rose, dehydro-assay is following:

Chromatographic condition and system suitability test high performance liquid chromatograph (U.S. Agilent company): comprise G1314A UV, visible light detecting device, G1314A binary pump, Agilent1100 chromatographic work station; Use octadecylsilane chemically bonded silica to be filling agent; With acetonitrile-water (65: 35) is moving phase; The detection wavelength is 225nm; Column temperature: 25 ℃.Number of theoretical plate calculates by the costunolide peak should be not less than 3000.

Get said Six-element banksia rose preparation 1g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 25ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 30 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, as banksia rose need testing solution; Other gets the costunolide reference substance with an amount of with the dehydro-reference substance; The accurate title, decide; Add methyl alcohol and process the mixed solution that every 1ml contains costunolide 40 μ g, dehydro-60 μ g, mix reference substance solution as costunolide and dehydro-.In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and processes banksia rose negative sample solution by the compound method of said banksia rose need testing solution." 2010 editions one appendix VI D of Chinese pharmacopoeia tests, and the above-mentioned three kinds of solution of accurate respectively absorption are 10 μ l respectively, and the injection liquid chromatograph is measured, and the result sees shown in accompanying drawing 4 according to high performance liquid chromatography.The every 1g of preparation contains the banksia rose and counts 2.43mg with costunolide and dehydro-total amount.

Experimental example 3: the assay-methodology of contained costunolide of the banksia rose and dehydro- Investigate

(1) instrument and reagent

Aglient 1100 high performance liquid chromatographs (U.S. Agilent company): G1314A UV, visible light detecting device, G1314A binary pump, Agilent1100 chromatographic work station;

SK8200HP ultrasonic cleaner (power 250W, frequency 40kHz) (Shanghai High Kudos Science Instrument Co., Ltd.);

Acetonitrile (chromatographically pure), methyl alcohol (analyzing pure);

Costunolide (lot number: 111524-201006), dehydro-(lot number: 111525-200907), all identify institute available from Chinese pharmaceutical biological product;

Six-element banksia rose preparation: provide by XiZang QiZheng Tibetan pharmaceuticals Co., Ltd.

(2) chromatographic condition

Octadecyl silane is a filling agent;

Chromatographic column: Kromasil E56526-C18 (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. for 4.6 * 250mm, 5.0 μ m);

Moving phase: acetonitrile-water (V: V=65: 35);

Flow velocity: 1.0ml/min;

Detect wavelength: 225nm;

Column temperature: 25 ℃.

(3) chromatographic system employment and suitability test (E & ST)

Under above-mentioned chromatographic condition, costunolide and dehydro-and all the other impurity peaks separating effects are better, and theoretical cam curve is calculated by the costunolide peak should be not less than 3000.

(4) preparation of reference substance solution, need testing solution, negative sample solution

The preparation of reference substance solution: get costunolide, the dehydro-reference substance is an amount of, accurately claim surely, add methyl alcohol and process the solution that every 1ml contains costunolide 40 μ g, dehydro-60 μ g, shake up, promptly get reference substance solution;

The preparation of need testing solution: get the about 1g of said Six-element banksia rose preparation, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 25ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 30 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate as need testing solution;

The preparation of negative sample solution: get the negative sample 1g that lacks banksia rose medicinal material, process negative sample solution with method, subsequent use.

(5) negative interference test

Accurate each the 10 μ l of reference substance solution, need testing solution and negative sample solution that draw; Inject liquid chromatograph; Can know by chromatogram, with the corresponding position of reference substance chromatographic peak retention time on, the chromatographic peak that need testing solution has identical retention time occurs; And negative sample solution does not have absorption at this wavelength, and is noiseless to the assay of costunolide in the preparation and dehydro-.The result sees chromatogram A, B, C shown in Figure 4.

(6) investigation of linear relationship

Get costunolide, the dehydro-reference substance is an amount of, accurate claims surely, adds methyl alcohol and processes the mixed solution that every 1ml contains costunolide 120 μ g and dehydro-170 μ g, shake up, as storing solution.The accurate absorption in above-mentioned stock solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 10ml to the 10ml volumetric flask; Be diluted to scale with methyl alcohol; Shake up, precision is measured 10 μ l and is injected liquid chromatograph respectively, measures peak area; Costunolide between 12～120 μ g/ml in the concentration of peak area and reference substance be good linear relationship, its regression equation is y=23.793x+10.253 (r=0.9997); Dehydro-between 17～170 μ g/ml in the concentration of peak area and reference substance be good linear relationship, its regression equation is y=14.982x+0.0062 (r=0.9996).The result sees table 1 and Fig. 5, table 2 and Fig. 6.

Table 1 costunolide contrast concentration and peak area result

Table 2 dehydro-contrast concentration and peak area result

(7) precision test

The same reference substance solution 10 μ l of accurate absorption, continuous sample introduction 5 times, the peak area of mensuration chromatographic peak, the result sees table 3.

Table 3 Precision test result

Test shows that precision is good, and RSD is respectively 0.61% and 1.34%.

(8) stability test

Accurate draw same need testing solution 10 μ l, respectively at 0,2,4,6, the 12h sample introduction, measure the peak area of chromatographic peak, the result sees table 4.

Table 4 stability test result

Test shows that the result is good at 0～12h internal stability, and RSD is respectively 0.79% and 1.55%.

(9) reappearance test

(lot number: 20100821) 5 parts, the preparation method handles according to test sample, distinguishes sample introduction, calculates content, total amount and the RSD of costunolide and dehydro-respectively, and the result sees table 5 to get said Six-element banksia rose preparation.

Table 5 reproducible test results

Test shows, 5 parts of mensuration of same lot sample article sampling, and reappearance is better as a result, and RSD is respectively 0.44%, 1.31% and 0.94%.

(10) recovery test

Get Six-element banksia rose preparation (lot number: 20100821, content is 2.656mg/g) the about 0.5g of powder of known content, accurate claim fixed; Add a certain amount of costunolide and dehydro-; Measure in accordance with the law,, try to achieve the content of costunolide and dehydro-respectively according to the chromatographic peak peak area value; Calculate recovery rate is measured the result and is seen table 6, table 7.

The average recovery test of table 6 costunolide

The result shows that this law recovery is good, and RSD is 1.26%.

The average recovery test of table 7 dehydro-

The result shows that this law recovery is good, RSD1.74%.

(11) mensuration of test sample

Get the about 1g of said Six-element banksia rose preparation, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 25ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 30 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter with 0.45 μ m filter membrane, get subsequent filtrate, promptly get need testing solution.The accurate need testing solution 10 μ l that draw measure in accordance with the law, calculate the content of costunolide and dehydro-, and the result sees table 8.

The assay result of costunolide and dehydro-in table 8 test sample

Mensuration result according to above-mentioned three multiple batches of test samples of producer can know; The every 1g of said Six-element banksia rose preparation contain the banksia rose in costunolide and dehydro-total amount between 1.352～2.656mg; Therefore, contain the banksia rose in the minimum tentative every 1g that limits the quantity of and to be not less than 1.30mg with costunolide and dehydro-total amount.

Following embodiment all can realize the effect of above-mentioned experimental example.

Test example 1: the detection of Six-element banksia rose ball

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element is ground into fine powder, sieves, and adds an amount of pill of water, promptly gets.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned Six-element banksia rose ball 2g, porphyrize adds ethyl acetate 20ml, and ultrasonic Extraction 30 minutes filters, and filtrating is concentrated into about 2ml, as need testing solution; Other gets the banksia rose, each 0.5g of seed of pomegranate control medicinal material, shines medicinal material solution in pairs with legal system; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution." each 10 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 5 μ l are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put respectively on same silica G plate according to thin-layered chromatography; With cyclohexane-methylene chloride-ethyl acetate (15: 5: 0.5) is developping agent, and thin layer plate was put in the expansion cylinder saturated 20 minutes, launches; Take out; Dry, spray is 5% vanillic aldehyde sulfuric acid solution with percent weight in volume, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned Six-element banksia rose ball 2g, porphyrize adds ethanol 20ml, and ultrasonic Extraction 30 minutes filters, and filtrating is concentrated into about 2ml, as need testing solution; Other gets emblic control medicinal material 0.5g, shines medicinal material solution in pairs with legal system; It is an amount of to get the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 1mg, as reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution." each 10 μ l of above-mentioned four kinds of solution are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put in same silica G F respectively according to thin-layered chromatography ₂₅₄On the plate, be developping agent with sherwood oil (60～90 ℃)-ethyl acetate-formic acid (6: 4: 0.25), thin layer plate was put in the expansion cylinder saturated 20 minutes, launched, and took out, and dried, and put under the ultraviolet lamp (254nm) and inspected.In the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color, and negative noiseless.

C. the assay of contained costunolide of the banksia rose and dehydro-

According to high performance liquid chromatography " 2010 editions one appendix VI D of Chinese pharmacopoeia

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water (65: 35) is moving phase; The detection wavelength is 225nm, and column temperature is 25 ℃, and number of theoretical plate calculates by the costunolide peak should be not less than 3000.

The preparation of reference substance solution: get the costunolide reference substance and the dehydro-reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains costunolide 40 μ g, dehydro-60 μ g, promptly get.

The preparation of need testing solution: get Six-element banksia rose ball 1g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 25ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 30 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get.

Determination method: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.

The every 1g of Six-element banksia rose ball contains the banksia rose with costunolide (C ₁₅H ₂₀O ₂) and dehydro-(C ₁₅H ₁₈O ₂) total amount count 2.43mg.

Test example 2: the detection of Six-element banksia rose hard shell capsules

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element is ground into fine powder, sieves, and mixing adds conventional auxiliary material by the pharmacy conventional method, processes acceptable clinically Six-element banksia rose hard shell capsules.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned Six-element banksia rose hard shell capsules 0.8g, porphyrize adds ethyl acetate 8ml, and ultrasonic Extraction 15 minutes filters, and filtrating is concentrated into about 0.5ml, as need testing solution; Other gets the banksia rose, each 0.2g of seed of pomegranate control medicinal material, shines medicinal material solution in pairs with legal system; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution." each 5 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 10 μ l are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put respectively on same silica G plate according to thin-layered chromatography; With cyclohexane-methylene chloride-ethyl acetate (12: 4: 0.3) is developping agent, and thin layer plate was put in the expansion cylinder saturated 30 minutes, launches; Take out; Dry, spray is 5% vanillic aldehyde sulfuric acid solution with percent weight in volume, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned Six-element banksia rose hard shell capsules 2g, porphyrize adds ethanol 20ml, and ultrasonic Extraction 30 minutes filters, and filtrating is concentrated into about 2ml, as need testing solution; Other gets emblic control medicinal material 0.5g, shines medicinal material solution in pairs with legal system; It is an amount of to get the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 1mg, as reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution." each 10 μ l of above-mentioned four kinds of solution are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put in same silica G F respectively according to thin-layered chromatography ₂₅₄On the plate, be developping agent with sherwood oil (60～90 ℃)-ethyl acetate-formic acid (6: 4: 0.25), thin layer plate was put in the expansion cylinder saturated 20 minutes, launched, and took out, and dried, and put under the ultraviolet lamp (254nm) and inspected.In the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color, and negative noiseless.

C. the assay of contained costunolide of the banksia rose and dehydro-

According to high performance liquid chromatography " 2010 editions one appendix VI D of Chinese pharmacopoeia

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water (64: 36) is moving phase; The detection wavelength is 225nm, and column temperature is 20 ℃, and number of theoretical plate calculates by the costunolide peak should be not less than 3000.

The preparation of reference substance solution: get the costunolide reference substance and the dehydro-reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains costunolide 30 μ g, dehydro-50 μ g, promptly get.

The preparation of need testing solution: get Six-element banksia rose hard shell capsules 0.2g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 15ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 20 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get.

Determination method: accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.

The every 1g of Six-element banksia rose hard shell capsules contains the banksia rose with costunolide (C ₁₅H ₂₀O ₂) and dehydro-(C ₁₅H ₁₈O ₂) total amount count 1.41mg.

Test example 3: the detection of Six-element banksia rose sheet

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element is ground into fine powder, sieves, and mixing adds conventional auxiliary material by the pharmacy conventional method, processes acceptable clinically Six-element banksia rose sheet.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned Six-element banksia rose sheet 1.5g, porphyrize adds ethyl acetate 15ml, and ultrasonic Extraction 20 minutes filters, and filtrating is concentrated into about 1ml, as need testing solution; Other gets the banksia rose, each 0.8g of seed of pomegranate control medicinal material, shines medicinal material solution in pairs with legal system; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution." each 15 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 4 μ l are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put respectively on same silica G plate according to thin-layered chromatography; With cyclohexane-methylene chloride-ethyl acetate (18: 6: 0.8) is developping agent; Launch, take out, dry; Spray is 5% vanillic aldehyde sulfuric acid solution with percent weight in volume, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned Six-element banksia rose sheet 0.8g, porphyrize adds ethanol 8ml, and ultrasonic Extraction 15 minutes filters, and filtrating is concentrated into about 0.5ml, as need testing solution; Other gets emblic control medicinal material 0.2g, shines medicinal material solution in pairs with legal system; It is an amount of to get the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 0.5mg, as reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution." each 5 μ l of above-mentioned four kinds of solution are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put in same silica G F respectively according to thin-layered chromatography ₂₅₄On the plate, be developping agent with sherwood oil (60～90 ℃)-ethyl acetate-formic acid (2: 3: 0.07), thin layer plate was put in the expansion cylinder saturated 30 minutes, launched, and took out, and dried, and put under the ultraviolet lamp (254nm) and inspected.In the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color, and negative noiseless.

C. the assay of contained costunolide of the banksia rose and dehydro-

According to high performance liquid chromatography " 2010 editions one appendix VI D of Chinese pharmacopoeia

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water (65: 35) is moving phase; The detection wavelength is 225nm, and column temperature is 20 ℃, and number of theoretical plate calculates by the costunolide peak should be not less than 3000.

The preparation of reference substance solution: get the costunolide reference substance and the dehydro-reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains costunolide 40 μ g, dehydro-60 μ g, promptly get.

The preparation of need testing solution: get Six-element banksia rose sheet 1g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 25ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 30 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get.

Determination method: accurate respectively reference substance solution and each 15 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.

The every 1g of Six-element banksia rose sheet contains the banksia rose with costunolide (C ₁₅H ₂₀O ₂) and dehydro-(C ₁₅H ₁₈O ₂) total amount count 2.15mg.

Test example 4: the detection of Six-element banksia rose particle

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element is ground into fine powder, sieves, and mixing adds conventional auxiliary material by the pharmacy conventional method, processes acceptable clinically Six-element banksia rose particle.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned Six-element banksia rose particle 3g, porphyrize adds ethyl acetate 30ml, and ultrasonic Extraction 45 minutes filters, and filtrating is concentrated into about 3ml, as need testing solution; Other gets the banksia rose, each 1.5g of seed of pomegranate control medicinal material, shines medicinal material solution in pairs with legal system; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution." each 10 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 5 μ l are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put respectively on same silica G plate according to thin-layered chromatography; With cyclohexane-methylene chloride-ethyl acetate (10: 3.5: 0.2) is developping agent, and thin layer plate was put in the expansion cylinder saturated 40 minutes, launches; Take out; Dry, spray is 5% vanillic aldehyde sulfuric acid solution with percent weight in volume, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned Six-element banksia rose particle 1.5g, porphyrize adds ethanol 15ml, and ultrasonic Extraction 20 minutes filters, and filtrating is concentrated into about 1ml, as need testing solution; Other gets emblic control medicinal material 0.8g, shines medicinal material solution in pairs with legal system; It is an amount of to get the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 1.5mg, as reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution." each 15 μ l of above-mentioned four kinds of solution are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put in same silica G F respectively according to thin-layered chromatography ₂₅₄On the plate, be developping agent, launch, take out, dry, put under the ultraviolet lamp (254nm) and inspect with sherwood oil (60～90 ℃)-ethyl acetate-formic acid (8: 5: 0.3).In the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color, and negative noiseless.

C. the assay of contained costunolide of the banksia rose and dehydro-

According to high performance liquid chromatography " 2010 editions one appendix VI D of Chinese pharmacopoeia

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water (66: 34) is moving phase; The detection wavelength is 225nm, and column temperature is 30 ℃, and number of theoretical plate calculates by the costunolide peak should be not less than 3000.

The preparation of reference substance solution: get the costunolide reference substance and the dehydro-reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains costunolide 60 μ g, dehydro-90 μ g, promptly get.

The preparation of need testing solution: get Six-element banksia rose particle 0.8g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 20ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 15 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get.

Determination method: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.

The every 1g of Six-element banksia rose particle contains the banksia rose with costunolide (C ₁₅H ₂₀O ₂) and dehydro-(C ₁₅H ₁₈O ₂) total amount count 1.94mg.

Test example 5: the detection of Six-element banksia rose soft capsule

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element is ground into fine powder, sieves, and mixing adds conventional auxiliary material by the pharmacy conventional method, processes acceptable clinically Six-element banksia rose soft capsule.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned Six-element banksia rose soft capsule 4.5g, porphyrize adds ethyl acetate 40ml, and ultrasonic Extraction 60 minutes filters, and filtrating is concentrated into about 4ml, as need testing solution; Other gets the banksia rose, each 2.5g of seed of pomegranate control medicinal material, shines medicinal material solution in pairs with legal system; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution." each 20 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 2 μ l are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put respectively on same silica G plate according to thin-layered chromatography; With cyclohexane-methylene chloride-ethyl acetate (20: 7.8: 0.6) is developping agent, and thin layer plate was put in the expansion cylinder saturated 20 minutes, launches; Take out; Dry, spray is 5% vanillic aldehyde sulfuric acid solution with percent weight in volume, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned Six-element banksia rose soft capsule 3g, porphyrize adds ethanol 30ml, and ultrasonic Extraction 45 minutes filters, and filtrating is concentrated into about 3ml, as need testing solution; Other gets emblic control medicinal material 1.5g, shines medicinal material solution in pairs with legal system; It is an amount of to get the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 2mg, as reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution." each 10 μ l of above-mentioned four kinds of solution are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put in same silica G F respectively according to thin-layered chromatography ₂₅₄On the plate, be developping agent with sherwood oil (60～90 ℃)-ethyl acetate-formic acid (3: 3: 0.1), thin layer plate was put in the expansion cylinder saturated 40 minutes, launched, and took out, and dried, and put under the ultraviolet lamp (254nm) and inspected.In the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color, and negative noiseless.

C. the assay of contained costunolide of the banksia rose and dehydro-

According to high performance liquid chromatography " 2010 editions one appendix VI D of Chinese pharmacopoeia

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water (63: 37) is moving phase; The detection wavelength is 225nm, and column temperature is 25 ℃, and number of theoretical plate calculates by the costunolide peak should be not less than 3000.

The preparation of reference substance solution: get the costunolide reference substance and the dehydro-reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains costunolide 120 μ g, dehydro-140 μ g, promptly get.

The preparation of need testing solution: get Six-element banksia rose soft capsule 1.5g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 35ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 30 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get.

Determination method: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.

The every 1g of Six-element banksia rose soft capsule contains the banksia rose with costunolide (C ₁₅H ₂₀O ₂) and dehydro-(C ₁₅H ₁₈O ₂) total amount count 2.23mg.

Test example 6: the detection that the Six-element banksia rose looses

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element is ground into fine powder, sieves, and mixing adds conventional auxiliary material by the pharmacy conventional method, processes the acceptable clinically Six-element banksia rose and looses.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get the diffusing 2g of the above-mentioned Six-element banksia rose, porphyrize adds ethyl acetate 20ml, and ultrasonic Extraction 30 minutes filters, and filtrating is concentrated into about 2ml, as need testing solution; Other gets the banksia rose, each 0.5g of seed of pomegranate control medicinal material, shines medicinal material solution in pairs with legal system; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution." each 10 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 5 μ l are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put respectively on same silica G plate according to thin-layered chromatography; With cyclohexane-methylene chloride-ethyl acetate (15: 5: 0.5) is developping agent; Launch, take out, dry; Spray is 5% vanillic aldehyde sulfuric acid solution with percent weight in volume, and it is clear that hot blast blows to the spot colour developing.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get the diffusing 4.5g of the above-mentioned Six-element banksia rose, porphyrize adds ethanol 40ml, and ultrasonic Extraction 60 minutes filters, and filtrating is concentrated into about 4ml, as need testing solution; Other gets emblic control medicinal material 2.5g, shines medicinal material solution in pairs with legal system; It is an amount of to get the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 2.5mg, as reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution." each 20 μ l of above-mentioned four kinds of solution are drawn in 2010 editions one appendix VI B test of Chinese pharmacopoeia, put in same silica G F respectively according to thin-layered chromatography ₂₅₄On the plate, be developping agent with sherwood oil (60～90 ℃)-ethyl acetate-formic acid (9: 2: 0.4), thin layer plate was put in the expansion cylinder saturated 20 minutes, launched, and took out, and dried, and put under the ultraviolet lamp (254nm) and inspected.In the test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color, and negative noiseless.

C. the assay of contained costunolide of the banksia rose and dehydro-

According to high performance liquid chromatography " 2010 editions one appendix VI D of Chinese pharmacopoeia

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With acetonitrile-water (67: 33) is moving phase; The detection wavelength is 225nm, and column temperature is 40 ℃, and number of theoretical plate calculates by the costunolide peak should be not less than 3000.

The preparation of reference substance solution: get the costunolide reference substance and the dehydro-reference substance is an amount of, accurately claim surely, add methyl alcohol and process the mixed solution that every 1ml contains costunolide 80 μ g, dehydro-170 μ g, promptly get.

The preparation of need testing solution: get the diffusing 2.5g of the Six-element banksia rose, porphyrize, the accurate title, decide, and puts in the tool plug conical flask; The accurate methyl alcohol 45ml that adds claims to decide weight, sonicated (power 250W, frequency 40kHz) 45 minutes; Put coldly, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get.

Determination method: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.

The diffusing every 1g of the Six-element banksia rose contains the banksia rose with costunolide (C ₁₅H ₂₀O ₂) and dehydro-(C ₁₅H ₁₈O ₂) total amount count 2.56mg.

Claims (15)

1. the detection method of a Six-element banksia rose preparation, said Six-element banksia rose preparation comprises the banksia rose, BAXIAGA, emblic, cardamom, seed of pomegranate and six kinds of medicinal ingredients of the Bi roots of grass; It is characterized in that this method is differentiated the banksia rose, seed of pomegranate, emblic and/or the Bi roots of grass in the said Six-element banksia rose preparation through thin-layered chromatography; Wherein:

(A) discriminating of the said banksia rose and seed of pomegranate may further comprise the steps:

(1) get said Six-element banksia rose preparation, add ethyl acetate, ultrasonic Extraction concentrates the back as the banksia rose, seed of pomegranate need testing solution with the gained extract;

(2) will go up the step gained banksia rose, seed of pomegranate need testing solution point sample in silica gel thin-layer plate, be that developping agent launches to differentiate with cyclohexane-methylene chloride-ethyl acetate; And/or

(B) discriminating of the said emblic and the Bi roots of grass may further comprise the steps:

(1 ') got said Six-element banksia rose preparation, adds ethanol, and ultrasonic Extraction concentrates the back as emblic, Bi roots of grass need testing solution with the gained extract;

(2 ') will be gone up step gained emblic, Bi roots of grass need testing solution point sample in silica gel thin-layer plate, be that developping agent launches to differentiate with petroleum ether-ethyl acetate-formic acid.

2. detection method according to claim 1, wherein:

Also comprise in the said step (1) and get the banksia rose, seed of pomegranate control medicinal material, process the banksia rose, seed of pomegranate control medicinal material solution respectively according to the compound method of the said banksia rose, seed of pomegranate need testing solution; Also comprising in the step (2) the banksia rose, seed of pomegranate control medicinal material solution point sample in silica gel thin-layer plate, is that developping agent launches with cyclohexane-methylene chloride-ethyl acetate; And/or

Also comprise in the said step (1 ') and get the emblic control medicinal material, add ethanol, ultrasonic Extraction concentrates the back as emblic control medicinal material solution with the gained extract; Get the pipering reference substance, add the methanol solution that methyl alcohol is processed pipering, as the pipering reference substance solution; Also comprising in the step (2 ') emblic control medicinal material solution, pipering reference substance solution point sample in silica gel thin-layer plate, is that developping agent launches with petroleum ether-ethyl acetate-formic acid.

3. detection method according to claim 1 and 2, wherein:

Comprise also in prescription ratio and preparation technology in the said step (1) that preparation does not contain the negative sample of the banksia rose, seed of pomegranate respectively, and processes the banksia rose, seed of pomegranate negative sample solution according to the compound method of the said banksia rose, seed of pomegranate need testing solution; Also comprising in the step (2) the banksia rose, seed of pomegranate negative sample solution point sample in silica gel thin-layer plate, is that developping agent launches with cyclohexane-methylene chloride-ethyl acetate; And/or

Comprise also in the said step (1 ') that in prescription ratio and preparation technology preparation does not contain the negative sample of emblic, the Bi roots of grass respectively, and processes emblic, Bi roots of grass negative sample solution according to the compound method of said emblic, Bi roots of grass need testing solution; Also comprising in the step (2 ') emblic, Bi roots of grass negative sample solution point sample in silica gel thin-layer plate, is that developping agent launches with petroleum ether-ethyl acetate-formic acid.

4. detection method according to claim 3, wherein:

Said step (1) is: get Six-element banksia rose preparation 0.5～5 weight portion, behind the porphyrize, add ethyl acetate 5～40 parts by volume, sonicated 10～60 minutes filters, and filtrating is concentrated into 0.5～5 parts by volume, as the banksia rose, seed of pomegranate need testing solution; Other gets each 0.1～3 weight portion of the banksia rose, seed of pomegranate control medicinal material, processes the banksia rose, seed of pomegranate control medicinal material solution respectively according to the preparation method of the said banksia rose, seed of pomegranate need testing solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the said banksia rose, seed of pomegranate need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL;

Preferably, said step (1) is: get Six-element banksia rose preparation 2 weight portions, behind the porphyrize, add ethyl acetate 20 parts by volume, sonicated 30 minutes filters, and filtrating is concentrated into 2 parts by volume, as the banksia rose, seed of pomegranate need testing solution; Other gets each 0.5 weight portion of the banksia rose, seed of pomegranate control medicinal material, processes the banksia rose, seed of pomegranate control medicinal material solution respectively according to the preparation method of the said banksia rose, seed of pomegranate need testing solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and processes the banksia rose, seed of pomegranate negative sample solution by the compound method of the said banksia rose, seed of pomegranate need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL.

5. according to each described detection method in the claim 1 to 4, wherein:

Said step (2) is: " sample solution that makes in the step (1) is drawn in an appendix VI of Chinese pharmacopoeia version in 2010 B test, and it is put respectively on same silica gel g thin-layer plate according to thin-layered chromatography; With cyclohexane-methylene chloride-ethyl acetate is developping agent, and thin layer plate is put in the expansion cylinder saturated, launches; Take out; Dry, spray is with the vanillic aldehyde sulfuric acid solution, and the colour developing of wind to spot is clear.

6. detection method according to claim 5, wherein:

Need testing solution in the sample solution of said absorption and negative sample solution are 5～20 μ l, control medicinal material solution is 1～10 μ l, and need testing solution in the sample solution of preferred said absorption and negative sample solution are 10 μ l, control medicinal material solution is 5 μ l; The volume ratio of said developping agent cyclohexane-methylene chloride-ethyl acetate is 8～22: 3～8: 0.1～1, and preferred volume ratio is 15: 5: 0.5; Said thin layer plate was put in the expansion cylinder saturated 0～40 minute, preferred saturated 20 minutes; Said vanillic aldehyde sulfuric acid solution percent weight in volume is 5%, and it is clear to blow to the spot colour developing with hot blast.

7. detection method according to claim 3, wherein:

Said step (1 ') is: get Six-element banksia rose preparation 0.5～5 weight portion, behind the porphyrize, add ethanol 5～40 parts by volume, sonicated 10～60 minutes filters, and filtrating is concentrated into 0.5～5 parts by volume, as emblic, Bi roots of grass need testing solution; Other gets emblic control medicinal material 0.1～3 weight portion, processes emblic control medicinal material solution according to the preparation method of said emblic, Bi roots of grass need testing solution; It is an amount of to get the pipering reference substance again, adds methyl alcohol and processes the solution that every 1ml contains pipering 0.5～3mg, as the pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of said emblic, Bi roots of grass need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL;

Preferably, said step (1 ') is: get Six-element banksia rose preparation 2 weight portions, behind the porphyrize, add ethanol 20 parts by volume, sonicated 30 minutes filters, and filtrating is concentrated into 2 parts by volume, as emblic, Bi roots of grass need testing solution; Other gets emblic control medicinal material 0.5 weight portion, processes emblic control medicinal material solution according to the preparation method of said emblic, Bi roots of grass need testing solution; It is an amount of to get the pipering reference substance again, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains pipering 1mg, as the pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and processes emblic, Bi roots of grass negative sample solution by the compound method of said emblic, Bi roots of grass need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL.

8. according to each described detection method in the claim 1 to 7, wherein:

Said step (2 ') is: " sample solution that makes in the step (1 ') is drawn in an appendix VI of Chinese pharmacopoeia version in 2010 B test, and it is put respectively on same silica GF254 thin layer plate according to thin-layered chromatography; With petroleum ether-ethyl acetate-formic acid is developping agent; Thin layer plate is put in the expansion cylinder saturated, launches, and takes out; Dry, under the 254nm ultraviolet lamp, inspect.

9. detection method according to claim 8, wherein:

The sample solution of said absorption is 5～20 μ l, preferably is 10 μ l; The volume ratio of said petroleum ether-ethyl acetate-formic acid is 1～10: 2～6: 0.05～0.5, and preferred volume ratio is 6: 4: 0.25, the boiling range of said sherwood oil is 60～90 ℃; Said thin layer plate was put in the expansion cylinder saturated 0～40 minute, preferred saturated 20 minutes.

10. according to each described detection method in the claim 1 to 9; It is characterized in that; Said detection method further comprises the content with contained costunolide of the banksia rose and/or dehydro-in the said Six-element banksia rose of the high effective liquid chromatography for measuring preparation, wherein:

The step of said high effective liquid chromatography for measuring comprises:

(1 ") preparation banksia rose need testing solution, and costunolide and/or dehydro-reference substance solution;

Step gained banksia rose need testing solution will be gone up in (2 "), and costunolide and/or dehydro-reference substance solution are injected liquid chromatograph and measured;

Preferably, also comprise preparation banksia rose negative sample solution in the said step (1 "); Comprise also in the said step (2 ") that banksia rose negative sample solution is injected liquid chromatograph to be measured.

11. detection method according to claim 10, wherein:

The chromatographic condition of said high effective liquid chromatography for measuring is: use octadecylsilane chemically bonded silica to be filling agent; With volume ratio is that 60～70: 30～40 acetonitrile-water is a moving phase; The detection wavelength is 225 ± 2nm; Column temperature: 20～40 ℃, number of theoretical plate calculates by the costunolide peak and is not less than 3000;

Preferably, the chromatographic condition of said high effective liquid chromatography for measuring is: use octadecylsilane chemically bonded silica to be filling agent; With volume ratio is that 65: 35 acetonitrile-water is a moving phase; The detection wavelength is 225nm; Column temperature: 25 ℃, number of theoretical plate calculates by the costunolide peak and is not less than 3000.

12. according to claim 10 or 11 described detection methods, wherein:

Said step (1 ") be: get Six-element banksia rose preparation 0.1～3 weight portion, behind the porphyrize, put in the tool plug conical flask, accurate methyl alcohol 10～50 parts by volume that add; claim to decide weight, at power 250W, sonicated is 15～45 minutes under the frequency 40kHz, puts cold; claim again to decide weight, supply the weight that subtracts mistake, shake up; filter, get subsequent filtrate, as banksia rose need testing solution with methyl alcohol; Precision takes by weighing the costunolide reference substance in addition and the dehydro-reference substance is an amount of; Add methyl alcohol and process the mixed solution that every 1ml contains costunolide 20～140 μ g and dehydro-40～200 μ g, mix reference substance solution as costunolide and dehydro-; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and processes banksia rose negative sample solution by the compound method of said banksia rose need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL;

Preferably, said step (1 ") is: get Six-element banksia rose preparation 1 weight portion, behind the porphyrize, put in the tool plug conical flask; accurate methyl alcohol 25 parts by volume that add, and claims decide weight, and at power 250W, sonicated is 30 minutes under the frequency 40kHz; put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methyl alcohol, shakes up; filtration, gets subsequent filtrate, as banksia rose need testing solution; Precision takes by weighing the costunolide reference substance with an amount of with the dehydro-reference substance in addition; The accurate title, decide; Add methyl alcohol and process the mixed solution that every 1ml contains costunolide 40 μ g and dehydro-60 μ g, mix reference substance solution as costunolide and dehydro-; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and processes banksia rose negative sample solution by the compound method of said banksia rose need testing solution; Wherein, when above-mentioned weight portion measured with g, parts by volume was in mL;

Preferably, said step (2 ") be: accurate respectively absorption need testing solution, reference substance solution, each 5～20 μ l of negative sample solution, preferred each 10 μ l inject liquid chromatograph, and mensuration promptly gets.

13. according to each described detection method in the claim 1 to 12, wherein:

The bulk drug of said Six-element banksia rose preparation is formed and is comprised: the banksia rose 200 weight portions, BAXIAGA 360 weight portions, emblic 500 weight portions, cardamom 80 weight portions, seed of pomegranate 400 weight portions and the Bi roots of grass 100 weight portions.

14. detection method according to claim 13, wherein:

The preparation method of said Six-element banksia rose preparation is: said bulk drug is ground into fine powder, sieves, mixing adds an amount of pill of water, promptly gets pill; Or said bulk drug is ground into fine powder, and sieving, mixing by the pharmacy conventional method, adds conventional auxiliary material, processes the oral formulations of accepting clinically: granule, pill, capsule, tablet or powder.

15. according to each described detection method in the claim 1 to 14, wherein, said Six-element banksia rose preparation is granule, pill, capsule, tablet or powder.

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